Immunofluorescence Analysis of Kidney Cubilin and Megalin

IF analyses were performed on serial sections of kidney biopsies. Samples were treated as previously described [92 (link)] and incubated overnight with primary antibody (sheep anti-human cubilin [R&D Systems, Minneapolis, MN, USA, cat. AF3700], rabbit anti-human megalin [LS-Bio, Seattle, WA, USA, cat. LS-B105]) diluted 1:100 in PBS 5% BSA at 4 °C. Sections were incubated with the appropriate fluorescent secondary antibody [92 (link)]. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Negative controls were run by omitting the primary antibody. Images were acquired with a DMI6000CS-TCS SP8 fluorescence microscope (Leica Microsystems, Milan, Italy) with a 20X/0.4 objective using a DFC365FX camera (Leica Microsystems, Milan, Italy) and analyzed with the LAS-AF software (Leica Microsystems, Milan, Italy).